Our perception of sound and our ability to balance our body in relation to head position is dependent on the proper functions of hair cells of the inner ear. Hair cells employ a transduction apparatus that detects mechanical deflections as small as the width of a hydrogen atom. Understanding how a hair cell achieves this remarkable sensitivity is fundamental to understanding the functions of the inner ear and to developing rational strategies for the amelioration of hearing and vestibular disorders. Mechanoelectrical transducer channels in hair cells use calcium (Ca) to regulate adaptation, the process whereby hair cells reset their sensitivity to small displacements. Hair bundles, the mechanical organelles, are exposed to endolymph, which has a bulk Ca concentration of approximately 30-100 uM. Because Ca blocks the transducer channels, the low concentrations of Ca in the endolymph, apparently, allow the channels to generate sufficient receptor potential while permitting enough Ca entry to support adaptation. Emerging preliminary data from our laboratory suggest that hair cells may express the plasma membrane Ca-ATPase (PMCA) at high density in the hair bundles. We hypothesize that hair bundle PMCA extrudes Ca to maintain sufficient extracellular Ca concentration in the microdomains of hair bundles in order to optimize the sensitivity of hair cells to displacement. At rest, a substantial amount of Ca enters hair cells through voltage-gated Ca channels expressed at the basolateral membrane. Ca is then shuttled to the hair bundles via diffusable protein buffers. This transcellular flow of Ca maintains the active Ca extrusion from the hair bundles to the endolymph, creating sufficient Ca concentration in the microdomains of the hair bundles. PMCA extrudes Ca in exchange for protons; consequently we expect a change in hair bundle pH from the activity of PMCA. To test this hypothesis, we will use biochemical analyses to determine the density and distribution of the candidate PMCA isoform/s in hair cells. Using electrophysiological techniques, we will characterize the pump current and determine the changes in Ca and proton concentration that result from the activity of the Ca pump. To meet all these goals, we will use hair cells from bullfrogs and mice. Finally, we will test our hypothesis directly by examining the sensitivity of hair cells in PMCA knockout mice. We predict that the sensitivity of hair cells from mice with a null mutation of candidate PMCA isoforms expressed in hair bundles will be severely compromised.